JP2005517403A - Human fetal bladder-derived epithelial cells - Google Patents

Abstract

The present invention discloses a substantially pure population of human bladder-derived epithelial cells and methods for isolating and culturing bladder-derived epithelial cells. In addition, several uses of human bladder-derived epithelial cells and cells that differentiate therefrom are disclosed herein. In another aspect of the invention, the invention creates a human tissue model of human fetal prostate epithelial cells by using a population of substantially pure human fetal bladder-derived epithelial cells to produce human fetal bladder-derived epithelial cells. To a non-human (mammalian recipient).

Description

(Refer to related applications)
This application claims priority to US Provisional Patent Application No. 60 / 357,035 (filed on Feb. 12, 2002) under 35 USC 119 (e), which is incorporated herein by reference. The whole is incorporated by reference.

(Technical field)
The present invention relates to the fields of developmental biology and cell biology, and more particularly to purified fetal human bladder-derived epithelial cells and fetal human bladder epithelial cell lines.

(Background technology)
The human bladder is essentially a two-layered organ consisting of the outer muscle wall and the highly specialized lumen epithelial lining. Differentiated bladder epithelium is characterized by the presence of urothelial plaques on the luminal surface of bladder superficial cells or umbrella cells. These plaques are characterized by a very unusual membrane structure (ie, an asymmetric unit membrane (AUM)) that is twice as thick as the cytoplasmic monolayer and has a monolayer on the luminal side. Thickening of the luminal layer is due to the presence of particles representing translucent tissue and is mainly composed of four transmembrane proteins: uroplakin (UP) Ia (27 kDa); UP Ib (28 kDa); UP II (15 kDa) and UP III (47 kDa). UP III is thought to play a role in the formation of urothelial glycocalyx and can interact with the cytoskeleton through the cytoplasmic moiety (Hu et al., 2000. J. Cell Biol. 151 (5). : 961-72).

  The developmental origin of the bladder is found in the endoderm tissue. The bladder arises from the total drainage space (both bladder and lower gastrointestinal tract are derived from the total space). As development progresses, the total drainage space is divided into the hindgut and urogenital sinus. The bladder muscle layer is derived from the adjacent mesenchyme by a signal from the bladder epithelium, but the identity of the signal is unknown.

  Among US men, bladder cancer is estimated to be about 2% of all malignancies and about 7% of all urinary tract malignancies, while women are about 1/3 of men It is. Furthermore, due to race, African Americans are almost twice as likely to be non-Spanish (adjusted by age). The American Cancer Society estimates that there will be more than 50,000 new bladder cancer patients in the following year, with an estimated 9500 deaths. For superficial (mild grade disease), chemotherapy is given intravesically (directly to the bladder) to collect the drug at the tumor site and remove any remaining tumor mass after resection. The Systemic chemotherapy and / or radiation is usually given in severe disease in combination with a radical cystectomy. However, in about 50% of severe patients, severe tumors recur sometime after cystectomy, so surgery to relieve the symptoms in these patients is rarely performed.

  In addition to race and sexual factors, chemical exposure is defined as a risk factor for bladder cancer. While the main risk factor for chemical exposure is smoking, occupational exposure (especially arylamines) is also an important risk factor.

  Many investigators have described the use of uroplakin antibodies for analysis of the differentiation stage of urothelial cells or diagnosis of metastatic bladder cancer (Yu et al., 1992. Epith. Cell Biol. 1: 4- b 12; Mol et al., 1993 Verh. Deutsch. Ges. Path. 77 and Moll et al., 1995. Am. J. Pathol.

  In vitro studies of urothelial cells typically utilize cell lines derived from bladder tumors, or primitive urothelial cells, but a limited number of normal urothelial tissue inducible cell lines Only has been reported (Christensen et al., 1984, Anticancer Res. 4: 319-38). International patent application WO 02/102997 discloses homozygous stem cells derived from unfertilized first meiotic diploid germ cells (which are said to be able to differentiate into various cell types). (Key et al., 1996, J Urol, 156 (6): 2073-8; and Owens et al., 1976, J. Natl. Cancer lnst. 56 (4): 843-9).

(Disclosure of the present invention)
In one aspect, the invention relates to a population of substantially pure human fetal urinary bladder-derived epithelial cells (with the ability to differentiate into bladder epithelium and / or prostate epithelium). Thus, a reference to the ability to differentiate into bladder epithelium or prostate epithelium refers to the ability to differentiate into any cell type. The cells can be cultured in serum-free medium and can have a cell surface that is substantially free of serum biomolecules. The human fetal urinary bladder-derived epithelial cells of the present invention express the H19 marker gene.

  In another aspect, the present invention provides a method of isolating the following substantially pure population of bladder-derived epithelial cells: (a) human fetal bladder-derived epithelial cells form a monolayer colony Maintaining sufficient and appropriate culture conditions; where human fetal bladder-derived epithelial cells migrate from microdissected bladder-derived epithelial cell sources to form monolayer colonies Is maintained in appropriate culture conditions sufficient to allow in a serum-free medium; where the bladder-derived epithelial cell origin is the culture condition sufficient to maintain the bladder-derived epithelial cells Wherein the serum-free medium contains nutrients including insulin, transferrin, α-tocopherol and aprotinin; and (b) acquires a substantially pure population of bladder-derived epithelial cells For, the step of subculturing the monolayer colonies. In some embodiments, the serum-free culture medium further comprises progesterone, keratinocyte growth factor (KGF) and heregulin (HRG).

  In another aspect, the present invention microdissects the origin of human fetal urinary bladder-derived epithelial cells; and, under sufficient culture conditions, maintains the bladder epithelium-derived epithelial cell origin to maintain the aforementioned urinary bladder-derived epithelial cells. Place in serum-free medium (where serum-free medium contains nutrients including insulin, transferrin, α-tocopherol and aprotinin); bladder-derived epithelium from bladder-derived epithelial cell origin to serum-free medium Maintaining appropriate culture conditions sufficient to allow cell migration; maintaining appropriate culture conditions sufficient to allow bladder-derived epithelial cells to form monolayer colonies; To obtain a substantially pure population of bladder-derived epithelial cells, a method of producing a substantially pure population of human fetal bladder-derived epithelial cells by subculturing the monolayer colony is provided. To. The medium used in this method may further contain progesterone, keratinocyte growth factor (KGF), and heregulin (HRG). Therefore, the human fetal urinary bladder-derived epithelial cells of the present invention are not derived from germ cells.

  In another aspect, the present invention provides a substantially pure population of human bladder-derived epithelial cells produced by the following steps: (a) human fetal bladder-derived epithelial cells form a monolayer colony Maintaining adequate culture conditions sufficient to allow for human fetal urinary bladder-derived epithelial cells from the microdissected bladder-derived epithelial cell origin to form a monolayer of colonies. Maintaining appropriate culture conditions sufficient to allow migration of bladder-derived epithelial cells into serum medium; where the origin of bladder-derived epithelial cells is sufficient to maintain bladder-derived epithelial cells Cultured in serum-free medium under various culture conditions, the serum-free medium contains nutrients including insulin, transferrin, α-tocopherol and aprotinin; and (b) derived from substantially pure bladder To acquire a population of endothelial cells, the step of subculturing the monolayer colonies. In some embodiments, the serum-free culture medium used in the step further comprises progesterone, keratinocyte growth factor (KGF) and heregulin (HRG).

  In a further aspect, the present invention comprises the step of microdissecting the origin of human fetal bladder-derived epithelial cells; serum-free origin of bladder-derived epithelial cells under sufficient culture conditions to maintain the aforementioned bladder-derived epithelial cells Placing in culture (where serum-free medium contains nutrients of insulin, transferrin, α-tocopherol and aprotinin); migration of bladder-derived epithelial cells from the origin of bladder-derived epithelial cells to serum-free culture Maintaining culture conditions sufficient to allow; maintaining culture conditions sufficient to allow bladder-derived epithelial cells to form monolayer colonies; and substantially pure bladder-derived epithelial cells To obtain a population of substantially pure human fetal urinary bladder-derived epithelial cells produced in the above-described subcultured monolayer colonies. The medium used in this method may further contain progesterone, keratinocyte growth factor (KGF), and heregulin (HRG).

  In yet another aspect, the present invention provides a method of maintaining a population of substantially pure human fetal bladder-derived epithelial cells (which have the ability to differentiate into bladder epithelium or prostate epithelium), and these human fetal bladder-derived epithelial cells A method for maintaining and culturing a population of cells, such that the cells retain the ability to differentiate while avoiding mitotic arrest.

  In yet another aspect, the present invention relates to a method of providing an immunogen source to a heterologous recipient, and the use of a population of substantially pure human fetal urinary bladder-derived epithelial cells as the immunogen. In some embodiments, the present invention provides a method of providing an immunogen source to a heterologous recipient and an effective amount of a number of humans of the present invention to induce an immune response in said recipient. The method includes the step of administering fetal bladder-derived epithelial cells to the aforementioned recipient.

  In yet another aspect, the present invention relates to a method of eliciting an immune response in a heterologous recipient, in order to induce an immune response in said recipient, an effective amount of a number of human fetal bladder-derived epithelial cells of the present invention. Administering to the recipient as described above.

  In yet another aspect, the present invention relates to a method for producing a population of human bladder epithelial cells differentiated from human fetal bladder-derived epithelial cells, and a non-human mammal capable of maintaining the proliferation of the aforementioned human fetal bladder-derived epithelial cells. The method includes the step of administering the human fetal urinary bladder-derived epithelial cells of the present invention to a position within an animal recipient (wherein the above-mentioned human fetal urinary bladder-derived epithelial cells are the above-mentioned human fetal urinary bladder-derived epithelial cells are converted into human urinary bladder epithelium) Recombinant ex vivo with mesenchymal tissue capable of differentiating into cells).

  In yet another aspect, the present invention provides a step of administering the human fetal urinary bladder-derived epithelial cells of the present invention to a location within a non-human mammal recipient capable of maintaining the growth of the aforementioned human fetal urinary bladder-derived epithelial cells. A group of human bladder epithelial cells differentiated from human fetal urinary bladder-derived epithelial cells produced by a process comprising (wherein said human fetal urinary bladder-derived epithelial cells are referred to as human fetal urinary bladder-derived epithelial cells Recombinant ex vivo with mesenchymal tissue capable of differentiating into cells).

  In yet another aspect, the present invention relates to a method for producing a population of human prostate epithelial cells differentiated from human fetal urinary bladder-derived epithelial cells, and a non-human mammal capable of maintaining the proliferation of the aforementioned human fetal urinary bladder-derived epithelial cells. The method includes the step of administering the human fetal urinary bladder-derived epithelial cells of the present invention to a position within an animal recipient (wherein the above-mentioned human fetal urinary bladder-derived epithelial cells are the above-mentioned human fetal urinary bladder-derived epithelial cells are converted into human prostate epithelial cells) Recombinant ex vivo with mesenchymal tissue capable of differentiating into cells).

  In yet another aspect, the present invention provides a step of administering the human fetal urinary bladder-derived epithelial cells of the present invention to a position in a non-human mammal recipient capable of supporting the growth of the aforementioned human fetal urinary bladder-derived epithelial cells. A group of human prostate epithelial cells differentiated from human fetal urinary bladder-derived epithelial cells produced by a process comprising (wherein said human fetal urinary bladder-derived epithelial cells are referred to as human fetal urinary bladder-derived epithelial cells Recombinant ex vivo with mesenchymal tissue capable of differentiating into cells).

  In yet another aspect of the invention, the invention creates a human tissue model of human fetal prostate epithelial cells by using a substantially pure population of human fetal bladder-derived epithelial cells, and human fetal bladder-derived epithelium. It relates to a method of administering a cell to a non-human (mammalian recipient).

  In yet another aspect of the present invention, the present invention creates a human bladder tissue model by using a substantially pure population of human fetal urinary bladder-derived epithelial cells, and proliferates the aforementioned human fetal urinary bladder-derived epithelial cells. The present invention relates to a method for administering human fetal urinary bladder-derived epithelial cells to a position within a non-human (mammalian recipient) capable of maintaining (wherein human fetal urinary bladder-derived epithelial cells are the aforementioned human fetal urinary bladder-derived epithelial cells) Can be recombined ex vivo with mesenchymal tissue capable of further differentiation into human bladder epithelial cells).

  In yet another aspect of the present invention, the present invention provides the human fetal urinary bladder-derived epithelial cells of the present invention at a position within a non-human mammal recipient capable of maintaining the proliferation of the aforementioned human fetal urinary bladder-derived epithelial cells. A human bladder tissue model produced by a process comprising the step of administering (wherein said human fetal bladder-derived epithelial cells are capable of differentiating said human fetal bladder-derived epithelial cells into human bladder epithelial cells) Recombined with leaf tissue and ex vivo).

  In yet another aspect of the invention, the invention creates a human tissue model of human fetal prostate epithelial cells by using a substantially pure population of human fetal bladder-derived epithelial cells, and human fetal bladder-derived epithelium. It relates to a method of administering cells to a non-human (mammalian recipient).

  In yet another aspect of the present invention, the present invention uses a population of substantially pure human fetal urinary bladder-derived epithelial cells to create a human prostate tissue model and to proliferate the aforementioned human fetal urinary bladder-derived epithelial cells. The present invention relates to a method for administering human fetal urinary bladder-derived epithelial cells to a position within a non-human (mammalian recipient) capable of maintaining (where human fetal urinary bladder-derived epithelial cells are the aforementioned human fetal urinary bladder-derived epithelial cells Can be recombined ex vivo with mesenchymal tissue capable of further differentiation into human prostate epithelial cells).

  In yet another aspect of the present invention, the present invention provides the human fetal urinary bladder-derived epithelial cells of the present invention at a position within a non-human mammal recipient capable of maintaining the proliferation of the aforementioned human fetal urinary bladder-derived epithelial cells. A human prostate tissue model produced by a process comprising the steps of: (wherein said human fetal bladder-derived epithelial cells have the ability to differentiate said human fetal bladder-derived epithelial cells into human prostate epithelial cells) Recombinant with some mesenchymal tissue and ex vivo).

  In another aspect of the invention, the invention relates to a method for providing cell therapy, whereby a substantially pure human fetal bladder-derived epithelial cell or a population of cells differentiated therefrom is introduced into a recipient. In some embodiments, the invention relates to a method of providing cell therapy to a recipient, comprising administering a number of human fetal urinary bladder-derived epithelial cells of the invention to said recipient, wherein said Human fetal urinary bladder-derived epithelial cells are first grown in serum-free medium and then administered to a location within the aforementioned recipient, which location is capable of maintaining the proliferation and differentiation of the bladder epithelial cells Position). In some embodiments, human fetal urinary bladder-derived epithelial cells of the invention are administered into the recipient's bladder lumen.

  In another aspect of the invention, the invention relates to a method for providing cell therapy, whereby a population of substantially pure human prostate epithelial cells differentiated from human fetal bladder-derived epithelial cells is introduced into a recipient. .

  In another aspect of the invention, the invention uses a substantially pure population of human fetal urinary bladder-derived epithelial cells as a source of drugs to be developed, as a source of human fetal urinary bladder epithelial cells or as a component of these cells To a method for providing a means for drug development.

  In another aspect of the invention, the invention uses a substantially pure population of human fetal urinary bladder-derived epithelial cells as the target for drugs to be developed as the source of human bladder epithelial cells or human prostate epithelial cells. And a method for providing a means for drug development.

  In another aspect of the invention, the invention provides a method of providing a source of nucleic acid or protein from these cells in the development of a bioassay, or the step of isolating nucleic acid or protein from human fetal bladder-derived epithelial cells. With respect to methods of providing an encompassing bioassay, the aforementioned nucleic acids or proteins are used as one or more major components in the bioassay. In another aspect, the present invention provides a source of human fetal urinary bladder-derived epithelial cells in the development of a bioassay or provides a bioassay comprising using human fetal urinary bladder-derived epithelial cells in a bioassay On how to do.

(Method for carrying out the present invention)
The following detailed description of the invention is provided to assist those skilled in the art in practicing the invention. Since modifications of the embodiments disclosed herein can be made by those skilled in the art without departing from the spirit of the invention, the detailed description of the invention should not be construed as limiting the invention. Absent. Throughout this disclosure, various publications, patents and published patent specifications are referenced by citation. The disclosures of these publications, patents, and published patents are hereby incorporated by reference in their entirety in this disclosure.

  The practice of the present invention uses conventional immunization techniques, molecular biology, microbiology, cell physiology, and recombinant DNA (which are within the purview of those skilled in the art) unless otherwise indicated (eg, , Molecular Cloning: Experimental Manual, 2nd edition, Sambrook et al. (1989); Current Protocols In Molecular Biology FM Ausubel et al. (1987); the series Methods A Inc. , MJ MacPherson, B. D. Hames and G. R. Taylor (1995), Antibodies, A Laboratory Man. al, Harlow and Lane, eds. (1988), Adult and Pediatric Urology, J.Gillenwater et al., eds. (2002), and Animal Cell Culture, see R.I.Freshney ed. (1987)).

(Definition)
As used herein and in the claims, the terms “urine bladder-derived epithelial cells”, “bladder-derived epithelial cells” and “bladder-derived cells” Refers to cells used interchangeably and derived from human fetal bladder epithelial tissue. These cells can divide and be passaged in vitro.

  An “antibody” is an immunoglobulin molecule that can bind to an antigen. As used herein, the term includes not only complete immunoglobulin molecules, but also anti-idiotypic antibodies, variants, fragments, fusion proteins, humanized, that contain antigen recognition sites that require specificity. Modified proteins, and modifications of immunoglobulin molecules.

“Monoclonal antibody” refers to a homogeneous antibody population, and the monoclonal antibody consists of amino acids (naturally occurring and nonnaturally occurring) that are involved in the selective binding of antigens. Monoclonal antibodies are very specific and are directed against a single antigenic site. The term “monoclonal antibody” does not include only complete and full-length monoclonal antibodies, but fragments thereof (eg, Fab, Fab ′, F (ab ′) ₂ , Fv), single chain (ScFv), Those variants, fusion proteins containing antibody portions, humanized monoclonal antibodies, chimerized monoclonal antibodies, and other optionally modified immunoglobulin molecular structures (binding to antigen recognition sites and antigens that require specificity) Including abilities). The source of the antibody or the method by which it is made is not limited to these (eg, hybridoma, phage selection, recombinant expression, transgenic animals, etc.).

  A “humanized” antibody refers to a molecule that has an antigen binding site derived from an immunoglobulin derived from a non-human species and the remaining structure is based on the structure and / or sequence of a human immunoglobulin. An antigen binding site can include either a complete variable domain fused onto a constant domain, or only a complementarity determining region (CDR) grafted onto an appropriate framework region in the variable domain.

  The term “antigen” is a molecule that can contain one or multiple epitopes to which an antibody can bind. An antigen is a substance that can have immunogenic properties (ie, induce an immune response). Antigens are considered a type of immunogen. As used herein, the term “antigen” refers to a full-length protein and peptide fragments thereof that contain one or multiple epitopes.

  The terms “surface antigen” and “cell surface antigen” are used interchangeably herein and refer to the plasma membrane components of a cell. These components include, but are not limited to, membrane-bound and peripheral membrane proteins, glycoproteins, polysaccharides, lipids and glycosyl phosphatidylinositol (GPI) binding proteins. A “membrane-bound protein” is a transmembrane protein that spans the lipid bilayer of the plasma membrane of a cell. A typical membrane-bound protein generally consists of at least one transmembrane segment containing hydrophobic amino acid residues. Peripheral membrane proteins do not extend into the hydrophobic interior of the lipid bilayer, and they are bound to the membrane surface by non-covalent binding interactions with other membrane proteins. A GPI binding protein is a protein maintained by a lipid tail that is inserted into the lipid bilayer at the cell surface.

  “Immunogen” refers to any substance that induces immune response. Substances that are immunogens are described as “immunogenic”. Induction of an immune response includes activation of a humoral response (eg, producing antibodies) or a cellular response (eg, priming cytotoxic T cells), an inflammatory response (eg, leukocyte recruitment), and Including but not limited to secretion of cytokines and lymphokines.

  The term “heterologous” when applied to a cell used for immunization or transplantation means that the cell is derived from a genetically distinct entity from the recipient. For example, a heterologous cell can be derived from a different species from the same species as the recipient, or from a different individual. Embryonic cells derived from an individual of one species are heterogeneous with an adult of the same species. “Heterologous” when applied to a recipient means that the recipient is genetically distinct from the source of this cell introduced into the recipient.

  “Explant” refers to bladder tissue removed from a human fetus. In general, explants are used as the origin of bladder-derived cells. Isolating cells from explants can be accomplished by several methods. One method involves placing explants of bladder epithelial tissue (either whole tissue or cut into smaller pieces) in the medium and allowing the epithelial cells to naturally migrate solid tissue masses into the medium. That is. Another method is to provide the tissue with enzymatic digestion or to provide mechanical force to cells away from the solid tissue.

  At least about 50% of the fetal bladder-derived epithelial cell surface, more preferably at least about 75% of the fetal bladder-derived epithelial cell surface, more preferably at least about 90% of the fetal bladder-derived epithelial cell surface, and most preferably at least fetal bladder-derived epithelial cell surface When about 95% of the epithelial cell surface does not have serum biomolecules derived from serum that are bound to the cell surface (so that the antigen or antibody binding site is bound or is due to an antibody or part of an antibody The antigenic recognition is not available) and the cell surface is “substantially free of serum biomolecules”. The cell surface can be determined by either microscopy or flow cytometry, which measures cell size. For example, various known sized synthetic beads are commonly used to adjust flow cytometry. A small amount of conditioning beads can be mixed with bladder-derived epithelial cells and the resulting population is analyzed by flow cytometry. The bladder-derived epithelial cells can then be compared to the size of the conditioning beads. Since the bead size is known, cell surface mass calculations can be achieved.

  As used herein, a “substantially pure” population of cells is at least about 85%, preferably at least about 90%, and more preferably more than about 95% (eg, 98%) of the cells of interest. % Or more).

  As used herein, “transplanted recombinant” refers to a binding unit of human fetal bladder-derived cells and mesenchymal tissue. The mesenchymal tissue can be of bladder origin or non-bladder origin (eg, bladder mesenchyme, seminal vesicle mesenchyme). The mesenchymal tissue can be from a heterologous species to the recipient. Mesenchymal tissue can also be derived from non-human species. The transplanted recombinant is a substrate, preferably a soft, biological substrate (eg, for 1 to 96 hours, more preferably about 6 to 48 hours, more preferably about 24 hours with overnight incubation). , Agar).

  As used herein, “serum” refers to the fluid phase of mammalian blood that remains after the blood has clotted.

  As used herein, “serum biomolecule” refers to a biological composition found in serum. Examples include, but are not limited to albumin, α1-globulin, α2-globulin, β-globulin, and γ-globulin. Serum biomolecules can include biological whole or partial compositions found naturally in serum or obtained from the processing and manipulation of serum.

  The term “mammal” or “mammal” refers to warm-blooded vertebrates, including but not limited to humans, mice, rats, rabbits, monkeys, recreational animals, and pets.

(Isolation and maintenance of human fetal bladder-derived epithelial cells)
The fetal bladder epithelial cells of the present invention are isolated from human fetal bladder tissue. The age of the fetus can be between about 6 weeks to about 40 weeks of pregnancy, preferably between about 8 weeks to about 30 weeks of pregnancy, more preferably between about 14 weeks to about 21 weeks of pregnancy, more preferably pregnancy. It can be about 16 weeks. The fetal bladder can be identified by gross anatomy, appearance, and location within the fetus. The bladder is located in the midline of the lower abdomen near the abdominal wall. In addition to identification by location and appearance, the bladder can be identified by attachment (on the upper surface of the bladder) to the allantoic tube, a remnant of the allantoic membrane known as the midline umbilical cord in the adult. Once identified, the fetal bladder is excised and preferably rinsed several times with phosphate buffered saline (PBS). Adjacent tissue and excess connective tissue are removed and dissected, and the bladder is rinsed again with PBS. The entire bladder is minced into approximately 1 mm cubes, suspended in basal medium, transferred to a centrifuge tube, and centrifuged to pellet the minced tissue. The supernatant is removed and the tissue is resuspended in additional basal medium and then transferred to the culture dish. Various basal media can be used to maintain the pH of the fluid within a range that promotes the survival of fetal bladder epithelial cells. Non-limiting examples include F12 / DMEM, Ham's F10 (Sigma), CMRL-1066, minimal basic medium (MEM, Sigma), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM, Sigma), OPTI-MEM® (GIBCO BRL) and Iscove's Modified Eagle's Medium (IMEM). Furthermore, Ham and Wallace (1979) Meth. Ens. 58:44, Barnes and Sato (1980) Anal. Biochem. , 102: 255, or Mather, J. et al. P. And Roberts, P .; E. (1998) Any basic culture described in "Introduction to Cell and Tissue Culture", Plenum Press, New York may also be used.

  Basal medium is added to the culture dish and the tissue is incubated under humidification (37 ° C.). For more optimal conditions to promote fetal bladder epithelial cell survival and proliferation, various nutrients can be added to supplement the basal medium (thus creating a “culture”). Examples include, but are not limited to insulin, transferrin, α-tocopherol (vitamin E), progesterone, heregulin (HRG), keratinocyte growth factor (KGF), and aprotinin. In preferred embodiments, the following amounts of nutrients are used to promote fetal bladder epithelial cell survival and proliferation: at least about 10 ng / ml insulin and no more than about 1 mg / ml insulin, more preferably about 10 μg. / Ml insulin; at least about 1 μg / ml transferrin and no more than about 100 μg / ml transferrin, more preferably about 10 μg / ml transferrin; at least about 0.1 μg / ml α-tocopherol and no more than about 1 mg / ml α-tocopherol, more preferably about 5 μg / ml α-tocopherol; at least about 0.3 nM progesterone and about 300 nM or less progesterone, more preferably about 3 nM progesterone; at least about 1 ng / ml KGF and Yo Less than about 100 ng / ml KGF, more preferably about 10 ng / ml KGF, at least about 0.5 nM HRG and less than about 500 nM HRG, more preferably about 5 nM HRG, and at least about 1 μg / ml Of aprotinin and no more than about 100 μg / ml aprotinin, more preferably about 25 μg / ml aprotinin. Antibiotics and / or antifungals (eg, gentamicin, penicillin, and / or streptomycin) can also be added to the medium, but antibiotics / antifungals are initially cultured (eg, the first 2-5 days) It is preferable to add only during. Human fetal bladder epithelial cells are preferably cultured in a serum-free medium.

  Fetal bladder epithelial cells migrate from the fetal bladder tissue to the medium in which the fetal bladder tissue is placed. In most cases, fibroblast type cells also migrate from the bladder tissue. Finely chopped tissue remnants (which do not adhere to the culture dish) flow through the medium and are cleared by medium change after a short period of culture (eg, 1-2 weeks). Fibroblast type cells are removed from the culture to produce a substantially pure population of fetal bladder-derived epithelial cells. Preferably, fibroblast type cells are removed by photoproteolytic digestion of the culture, preferably in the presence of a calcium chelator such as EDTA. Cultures are observed microscopically during proteolytic treatment using, for example, trypsin-EDTA solution (eg, GIBCO BRL product no. 25300-054) to detect fibroblast-type cell detachment. (Preferably using an inverted phase contrast microscope). When fibroblast type cells are detached, they are removed from the culture by aspiration. The remaining cells in the culture are rinsed with PBS and cultured in fresh medium.

  Fetal bladder epithelial cells can be grown either in uncoated tissue culture vessels (eg, flasks, plates, etc.) or tissue culture vessels coated with different substrates. Non-limiting examples of substrates that can be used include fibronectin, laminin, collagen, polylysine, nitrocellulose, nylon, and polytetrafluoroethylene. The size of the tissue culture container is proportional to the amount of bladder epithelial tissue placed in the container. One skilled in the art can determine the correct size tissue culture container by stepwise growth of fetal bladder tissue placed in the tissue culture container. When fetal bladder tissue is first placed in a tissue culture container, the medium is generally transparent in overall turbidity. As the cells migrate and move away from the bladder tissue fragment, the medium becomes more opaque and more turbid. When the medium becomes very turbid, additional medium is added to the tissue culture vessel to supplement the nutrients consumed by the bladder-derived cells by adding more fresh medium or by completely changing the medium. Placed in. Additionally or alternatively, if the medium becomes cloudy, a small amount of cells can be removed from the tissue culture vessel and the cell viability can be checked using, for example, trypan blue staining. Tissue culture vessels that are overfilled with too many cells begin to see a decrease in cell viability.

  In general, continuous culture of fetal bladder-derived epithelial cells involves the transfer of cells to one or more new culture vessels. Preferably, such a transition is performed (eg, as indicated by reduced cell viability) before the culture vessel is overloaded with cells. The cells can be transferred to other large sized containers (eg, larger volumes) to accommodate increasing cell volumes. In addition, the cells can be “split” into several separate tissue culture vessels with fresh media (known as “passaging”). In this way, a substantially pure population of fetal urinary bladder-derived epithelial cells can be obtained and expanded.

  Removal of the cells from the tissue culture vessel is preferably accomplished by enzymatic treatment to detach the cells from the surface of the plastic tissue culture vessel. In a more preferred embodiment, an enzyme such as collagenase dispase is used in an effective amount to detach fetal bladder epithelial cells from the side of the tissue culture flask. An effective amount is at least about 10% by weight collagenase dispase, more preferably at least about 1% by weight collagenase dispase, and most preferably at least about 0.1% by weight collagenase dispase. After detachment of the cells from the surface of the tissue culture vessel, the enzyme is washed with basal medium (preferably the culture medium disclosed herein) and the cells are culture medium (preferably disclosed herein) Placed in a new culture vessel with In a preferred embodiment, keratinocyte growth factor (KGF) (also referred to as fibroblast growth factor 7 (FGF7)) and heregulin (HRG) are used as growth factors in medium supplements for the growth and longevity of bladder-derived epithelial cells. Is done.

  The frequency of feeding fetal bladder-derived epithelial cells depends on the nutrient metabolic rate of the cells. If the nutrient metabolic rate is high, the cells frequently require nutritional supplementation. In general, when cells metabolize nutrients in the medium, the acidity of the medium increases. Some cultures (e.g. RPMI-1640, DMEM, EMEM, etc.) contain pH sensitive dyes that show acidity, and as a result, the medium changes color when it becomes acidic. Culture media can then be added to bring the existing medium acidic to the acidity that sustains life and promotes cell growth. Alternatively, a small portion of the cells can be removed from the tissue culture vessel and cell viability can be assessed, for example, with trypan blue staining. If the culture is metabolized, cell viability is low (eg, less than 50%). To promote the survival and proliferation of fetal urinary bladder-derived epithelial cells, the preferred frequency of supplementation is about twice per week. The fetal urinary bladder-derived epithelial cells of the present invention can be passaged multiple times (up to a maximum of 12 times) in the absence of arrest.

(Characteristics of fetal bladder-derived epithelial cells)
The population of fetal urinary bladder-derived epithelial cells of the present invention, isolated by the methods disclosed herein, has several defined characteristics. Identification of fetal bladder-derived epithelial cells can be accomplished by morphology or specific markers, or a combination of both techniques.

  The morphology of fetal bladder-derived epithelial cells is characterized by the properties of transitional epithelial cells. The cells proliferate, clustering epithelial cell colonies. Since it is a transitional epithelial tissue, the morphology changes somewhat. In the middle are closely polygonal epithelial cells, cells that have become elongated due to the slimming process, and other cells morphologically. The elongated cells formed cell clumps. Closely polygonal cells express less urothelial cell differentiation markers (eg, cytokeratin 7 and cytokeratin 19, and uroplakin III) or do not express urothelial cell differentiation markers. Cells represented bladder epithelial cells with reduced differentiation. Morphologically expanded elongated and round cells displayed more differentiated properties.

  Markers that can be used to detect fetal bladder-derived epithelial cells are cytokeratin (CK) 1, cytokeratin 5, cytokeratin 6, cytokeratin 7, cytokeratin 8, cytokeratin 10 on the surface of fetal bladder-derived epithelial cells. , Cytokeratin 11, cytokeratin 13, cytokeratin 15, cytokeratin 16, cytokeratin 18, and cytokeratin 19 and uroplakin (eg, uroplakin Ia, uroplakin Ib, uroplakin II, and uroplakin III), but are not limited to these Not. These cell surface markers are evaluated by using antibodies specific for CK and uroplakin. Examples of antibodies that may be used include, but are not limited to: anti-cytokeratin CK-7 (Zymed 18-0234 lot 00460118), CK-19 (NCL-CK19 mouse monoclonal Batch 100902) and anti-uroplakin Antibody (mouse anti-uroplakin III clone AUI, mIgG1 lot number 105300a catalog number RDI-PR0651108, RDI). Anti-CK and anti-uroplakin antibodies can be used either in immunohistochemistry or by flow cytometry, either directly or indirectly from bladder-derived epithelial cells.

  Fetal bladder-derived epithelial cells are also stained with an anti-α-actin antibody (DAKOA / S Clone 1A4 Code M0851), which is a smooth muscle actin marker. This result is negative, indicating that cultured fetal bladder-derived epithelial cells do not contain any smooth muscle cells. Yet another marker that can be used in additional properties is the H19 imprint gene, as shown in Example 3.

(Use of fetal bladder-derived epithelial cells)
(Immunogen)
Use in fetal bladder-derived epithelial cells is an immunogen. As disclosed in the present invention, unique serum-free culture conditions allow the cell surface of fetal bladder-derived epithelial cells to remain free of serum proteins or serum biomolecules that can be bound to the surface. The potential problem of antigenic sites “masked” by binding by serum biomolecules is avoided by using the disclosed serum-free isolation and using culture techniques. Thus, a panel of antibodies can be made with newly available antigens that are “covered” when using culture conditions that include serum.

  Fetal bladder-derived epithelial cells isolated and cultured using the methods disclosed herein can be used as immunogens administered to xenogeneic recipients. Methods of administering fetal urinary bladder-derived cells as an immunogen to a heterologous recipient include, but are not limited to: immunization, administration to membranes by direct contact (eg, application or abrasion device), aerosols Administration to the mucosa and oral administration. As is well known in the art, immunity can be either passive or active immunity. Immunization methods can occur through different routes including, but not limited to, intraperitoneal injection, intradermal injection, and local injection. The immunized subject may include a mammal such as a mouse. In general, immune pathways and immunization schedules are established conventional techniques in antibody stimulation and production. When a mouse is used in this embodiment, any mammalian subject, including humans, or antibody-producing cells therefrom, can serve as a basis for the production of a mammalian hybridoma cell line. It can be manipulated according to the process. Typically, mice are inoculated intraperitoneally with an immunogenic amount of fetal urinary bladder-derived cells and then boosted with the same amount of immunogen. Optionally, cells grown in a non-biological membrane matrix are surgically implanted into the peritoneal cavity of the host mammal. Lymphoid cells (preferably mouse-derived splenic lymphoid cells) are harvested a few days after the last boost and cell suspensions are prepared from these for use in cell fusion.

  Hybridomas are described in Kohler, B and Milstein, C .; (1975) Nature 256: 495-497 (corrected in Buck, DW et al. (1982) In Vitro, 18: 377-381), using systemic somatic cell hybridization techniques, lymphocytes and immortalized myeloma. Prepared from cells. Available myeloma cell lines include, but are not limited to, X63-Ag8.653, and are available from the Silk Institute, Cell Distribution Center, San Diego, Calif. , Cell lines from USA can be used for hybridization. This technique fuses myeloma cells and lymphoid cells using a fusion source (eg, polyethylene glycol) or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium such as HAT medium to remove unhybridized parent cells. Any of the media described herein can be used to culture hybridomas that secrete monoclonal antibodies. As another alternative to cell fusion technology, EBV immortalized B cells are used to produce the monoclonal antibodies of the invention. If desired, the hybridoma is grown and passaged, and the supernatant is subjected to conventional immunoassay procedures (eg, radioisotope labeled immunoassay, enzyme immunoassay, or fluorescent immunoassay). To assay for anti-immunogenic activity.

  Hybridomas producing such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from media or body fluids, if desired, by conventional immunoglobulin purification procedures such as sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration. If undesired activity is present, for example, by attaching an adsorbent made with the immunogen to the solid phase, pouring the preparation thereon, and eluting or dissociating the desired antibody from the immunogen. Can be removed.

  Thus, a new panel of antibodies against cell surface antigens specific for fetal bladder-derived epithelial cells can be created using the fetal bladder-derived epithelial cells of the present invention. A population of monoclonal antibodies that bind to representative cell surface antigens of human fetal urinary bladder-derived epithelial cells can also be generated using the methods described in PCT WO 00/37503. Once monoclonal antibodies against cell surface antigens on fetal urinary bladder-derived epithelial cells are made by the methods disclosed herein, the antibodies have several uses.

  For purposes of making a recombinant or humanized antibody, the antibody can be sequenced and cloned. Other uses of specific antibodies for bladder-derived epithelial cells include biological testing or purification (eg, isolating fetal bladder-derived epithelial cells by flow cytometry or panning), therapeutic uses (eg, to target cells) Promotes or inhibits cell growth by binding antibodies, or promotes or blocks cell mass growth by binding antibodies to target cells, biological markers (eg, from other fetal bladders) Cell identification), and clinical diagnosis (eg, identification of cancerous bladder epithelial cells).

(Drug discovery)
Another use of human fetal urinary bladder-derived epithelial cells (or differentiated cells derived from human fetal urinary bladder-derived epithelial cells) relates to drug discovery. Since the human fetal urinary bladder-derived epithelial cell population has not been previously isolated and cultured in the disclosed methods, the fetal urinary bladder-derived epithelial cell population has not been previously discovered or characterized. Can secrete no protein. Previous culture methods using serum can inhibit protein secretion. Alternatively, proteins can change function, conformation, or activity when secreted and interacting with serum biomolecules. Proteins secreted by fetal urinary bladder-derived epithelial cells have minimal damage from serum biomolecules, and as a result can be more physiological and topological. Thus, proteins secreted by fetal bladder-derived epithelial cells can be used as targets for drug development. In one embodiment, the drug can be made to target specific proteins on fetal bladder-derived epithelial cells and / or cells differentiated therefrom in vivo. Drug binding can promote differentiation of bladder cells into fully differentiated bladder epithelial cells. This approach can be used when neoplasia of bladder epithelial cells is desired, for example, after radiation therapy involving the bladder or after intravesical chemotherapy (which can damage or destroy the bladder epithelium).

  In yet another application, human fetal bladder-derived epithelial cells (or differentiated cells derived from human fetal bladder-derived epithelial cells) are used to develop or discover small molecules that interact with bladder-derived epithelial cells. obtain. These small molecules can be synthetic or natural and can be used to inhibit or promote proliferation and / or differentiation of bladder-derived epithelial cells.

  In yet another application, human fetal bladder-derived epithelial cells (or differentiated cells derived therefrom) can be used as a source of tissue-specific components in the pharmaceutical development of one or more drugs, and these Are used as targets for drugs under development.

(Cell therapy)
In another application, fetal bladder-derived epithelial cells are used for cell therapy. Transplantation of fetal bladder-derived epithelial cells is an example of cell therapy. To carry out this application, fetal bladder-derived epithelial cells are isolated and cultured in serum-free (a defined culture using the disclosed method). As disclosed herein, cell populations can be passaged to expand the number of fetal bladder-derived epithelial cells available for transplantation. The fetal urinary bladder-derived epithelial cells can then be administered to the recipient and repopulate the luminal surface of the bladder. Alternatively, fetal bladder-derived epithelial cells can be used as a cellular carrier for gene therapy (where fetal bladder-derived epithelial cells are transfected with one or more genes and then administered to the recipient (preferably, Intravesical administration)). In another embodiment, fetal bladder-derived epithelial cells are used in devices (including cells and restricting access from other cells to limit the immune system response) (eg, Therapete®). ).

  The plasticity of the fetal urinary bladder-derived epithelial cells of the present invention can also be exploited in the context of the prostate. Thus, fetal bladder-derived epithelial cells can be transplanted into individual prostates to repopulate or enlarge the prostate epithelium. Fetal bladder-derived epithelial cells can also be administered by administration of genetically modified fetal bladder-derived epithelial cells (eg, after transfection of one or more genes into fetal bladder-derived epithelial cells, for delivery to the prostate. Can be used as a cellular carrier for gene therapy.

(Human tissue model)
Another use of fetal urinary bladder-derived epithelial cells is to create human tissue models in non-human mammals. Fetal bladder-derived epithelial cells are placed on mesenchymal tissue to form transplanted recombinants. The mesenchymal tissue can be either bladder-derived tissue or non-bladder-derived tissue, and can be from a different species from which fetal bladder-derived epithelial cells are isolated. In an example, human fetal bladder-derived epithelial cells are placed on rat bladder mesenchymal tissue to form transplanted recombinants. Another exemplary transplant recombinant is fetal bladder-derived epithelial cells placed on the prostate mesenchyme (eg, rat seminal vesicle mesenchyme). One of skill in the art will first be able to isolate human fetal urinary bladder-derived epithelial cells using the methods disclosed herein, and then in a step-by-step manner by combining with mesenchymal tissue from different organs. The optimal combination can be determined. In some embodiments, different species (eg, rats) are used as a source of mesenchymal tissue in combination with human fetal bladder-derived epithelial cells. The use of heterologous species allows human specific markers to be used to determine the identity of differentiated fetal bladder-derived cells. When rat mesenchymal tissue is used, the likelihood of false positives is reduced.

  The transplanted recombinant comprising the fetal urinary bladder-derived epithelial cell region located in the mesenchymal tissue is preferably cultured on a soft substrate (eg, agar) for about half a day to about 3 days, more preferably about 1 day, and then Placed under the kidney capsule of the recipient mammal. Possible recipient mammals include, but are not limited to, mice and rats. Typically, in a transplant situation, donor tissue is vulnerable to attack by the recipient's immune system. Several techniques can be used to mitigate graft rejection. One method is to irradiate the recipient with a sublethal dose of radiation to destroy immune cells that can attack the graft. Another method is to give the recipient a cyclosporine or other T cell immunosuppressant. In using the mouse as a recipient mammal, various methods are possible to alleviate graft rejection. One such method is the use of immunodeficient mice (nude or severe combined immunodeficiency or SCID).

  In the examples, the human fetal urinary bladder-derived epithelial cell region is located on rat bladder mesenchymal tissue or rat seminal vesicle mesenchyme and below the kidney capsule of immunodeficient mice. Before the graft is expanded and analyzed for fetal urinary bladder-derived epithelial cell differentiation, the transplanted recombinant is between about 1 week and about 52 weeks, preferably between about 5 weeks and about 40 weeks, more preferably Remain in the recipient for between about 6 weeks and about 10 weeks, and more preferably for about 8 weeks. In some cases, a small transplant is required for analysis. As disclosed herein, fetal bladder-derived epithelial cells and markers specific for cells derived therefrom can be utilized in immunohistological analysis. In addition, combinations of one or more of these markers can be used in combination with cell morphology to determine the effect of transplantation.

  In one embodiment, the human fetal bladder-derived model is created in SCID (severe combined immunodeficiency) mice. This fetal urinary bladder-derived model can be created by utilizing isolated and cultured human fetal urinary bladder-derived epithelial cells using the methods disclosed herein and creating transplanted recombinants Can be made by using human fetal urinary bladder-derived epithelial cells. The transplanted recombinant is then placed under the kidney capsule of the mouse. About 1-10 weeks after transplantation under the kidney capsule, preferably about 6-8 weeks, the graft or a portion thereof is collected and analyzed by immunohistology. Cell surface markers on fetal bladder-derived epithelial cells that can be used are CK1, CK5, CK6, CK7, CK8, CK10, CK11, CK13, CK15, CK16, CK18 and CK19, prostate specific antigen (PSA), acidic alkali Including, but not limited to, phosphatase, uroplakin Ia, uroplakin Ib, uroplakin II and uroplakin III, and the H19 imprint gene. Where PSA antibodies are useful for analyzing prostate tissue model systems, the anti-CK antibodies or anti-uroplatin antibodies disclosed herein are used to analyze the effects of bladder tissue model systems. Another way to evaluate the outcome of fetal bladder-derived epithelial cell differentiation is by morphology. Bladder-derived epithelial cells form a transitional epithelium and always form a cavity structure alongside the bladder epithelium when recombined with bladder mesenchymal tissue. Bladder-derived epithelial cells recombined with seminal vesicle mesenchyme take on typical prostate epithelial morphology. For a more complete assessment, morphology can be coupled to cell surface markers.

  Through these methods, the present invention also provides a method for producing a population of differentiated human bladder epithelial cells and human prostate epithelial cells.

(Bioassay)
The human fetal urinary bladder-derived epithelial cells (or cells differentiated therefrom) and components of these cells disclosed herein can be used in various bioassays. In one application, bladder-derived epithelial cells are used to determine which biological factors are required for differentiation. By using bladder-derived epithelial cells in a step-wise manner in a combination of different biological compounds (eg, hormones, specific growth factors, etc.), one or more specific biological compounds can be Or it can be found to induce differentiation in prostate cells.

  The invention also provides a method for providing a source of nucleic acid or protein for a bioassay comprising the step of isolating nucleic acid or protein derived from human fetal urinary bladder-derived epithelial cells, and as one or more major components in the bioassay, Methods of using the nucleic acids or proteins are provided. Other uses of bioassays for bladder-derived epithelial cells are differential-display methods (eg, mRNA differential display methods) and protein-protein interactions using secreted proteins from bladder-derived epithelial cells. Protein-protein interactions can be determined by techniques such as, for example, a yeast two-hybrid system. Proteins derived from bladder-derived epithelial cells can be used to identify other unknown proteins or other cell types that interact with bladder-derived epithelial cells. These unknown proteins can be one or more of the following: growth factors, hormones, enzymes, transcription factors, translation factors and tumor suppressors. Bioassays for bladder-derived epithelial cells and the protein-protein interactions they form, as well as the effects of protein-protein or uniform cell-cell contacts, how surrounding tissue such as mesenchymal tissue is bladder It can be used to determine whether it contributes to the differentiation of derived epithelial cells.

  The following examples provide a detailed description of the isolation, characterization, and use of fetal bladder-derived epithelial cells. These examples are not intended to limit the invention in any way.

(Example 1 Isolation of fetal bladder-derived epithelial cells)
Human fetal bladder tissue (gestational age 14-21 weeks) was obtained from Advanced Bioscience Research at Alameda, California. As soon as the tissues arrived, they were rinsed (3 times) with 20 ml of cold PBS. Excess connective tissue was removed from the bladder, rinsed twice more with cold PBS, then dissected under a microscope with a razor blade or scissors and cut into small segments (chopped to approximately 1 mm).

  Suspend minced tissue in 10 ml OPTI-MEM® (Invitrogen Life Technologies), then use plastic pipette (precoated with bovine serum albumin to minimize tissue binding to the pipette wall) And transferred to a 15 ml centrifuge tube. To pellet the minced tissue, the minced tissue was centrifuged at 1000 rpm for 5 minutes in Heraeus Megafuge 2.0 (Cat # 75003485). The supernatant was aspirated and the pellet was resuspended with 6 ml of fresh OPTI-MEM®.

The tissue suspension was transferred and dispensed into 6-well tissue culture treated 6-well tissue culture plates. In addition, 3 ml of culture solution (10 μg / ml insulin, 10 μg / ml transferrin, 5 μg / ml α-tocopherol (Sigma catalog number T3251), 25 μg / ml aprotinin, 3 nM progesterone, 10 ng / ml KGF, 5 nM HRG, 100 μg / ml gentamicin OPTI-MEM®) was added to the wells and the plates were then incubated at 37 ° C. in a humidified incubator with 95% air (5% CO ₂ atmosphere). Gentamicin was not used after the first 2 days of culture.

  Fibroblasts were removed from the culture by limited proteolysis. Cultures were trypsinized briefly with 0.5 ml 0.05% EDTA-trypsin (Gibco BRL) and observed under a phase contrast microscope until the fibroblasts shrunk. The plate was shaken thoroughly and the fibroblasts were detached from the plate. When fibroblasts were detached from the substrate, the fibroblasts were removed by aspirating the medium and the cells were detached. The culture was rinsed twice with 5 ml of PBS and then fed with fresh media.

  The resulting purified fetal bladder-derived epithelial cell population was passaged up to 12 times. Cells were detached from the incubator by trypsinization (1 ml 0.05% trypsin-EDTA, Invitrogen Life Technologies catalog number 25300-054) or collagenase / dispase (3 mg / ml) (Roche Molecular Biochemicals (catalog number 269638). Exfoliated cells were collected, pelleted (1000 rpm for 5 minutes), washed twice with basal medium, resuspended with fresh media, and replated.

Example 2 Characterization of immunohistochemistry
Monolayer fetal urinary bladder-derived epithelial cells cultured in 4-well chamber slides were washed 3 times with cold PBS and fixed with 100% reagent alcohol at −20 ° C. for 5 minutes. The fixed slide was then air dried overnight. The cells are treated with blocking buffer (PBS containing 5% goat serum) for about 1 hour, with primary antibody overnight, and with peroxidase-binding F (ab) ₂ fragment goat anti-mouse IgG + IgM (H + L) for about 1 hour. Incubated continuously. During these steps, cells were washed 3 times with PBS (5 min / wash). The primary antibodies used were cytokeratin 7 and cytokeratin 19, uroplakin III and α-actin at dilutions recommended by the vendor. In order to visualize the staining of the cells with antibodies, fetal bladder-derived epithelial cells were incubated with the peroxidase substrate DAB / H ₂ O ₂ prepared from Sigma.

  Some cells were positive for uroplakin III and cytokeratin 7 and cytokeratin 19. These cells were usually located towards the edge of the epithelial colony. A smaller number of differentiated cells located in the center of the epithelial colony were negative for these markers. Cells in culture are not positive for α-actin. Positive staining for uroplakin III (a very specific marker for bladder epithelium) indicates that the cells are bladder epithelium, but the lack of staining with α-actin indicates that the culture is contaminated with smooth muscle cells Indicates not.

(Example 3 Detection of Fetal Bladder Cell Molecular Marker Hl9 Imprinted mRNA Gene)
The H19 imprinted mRNA gene is characterized as a marker that is specifically expressed in fetal human bladder cells, but normal human adult bladder cells (Elkin et al., 1995, FEBS Lett. 374 (1): 57-61; Cooper Et al., 1996, J. Urol. 155 (6): 2120-27). To detect Hl9 mRNA, two passages of homotypic fetal urinary bladder-derived epithelial cell cultures (about 10 ⁵ cells each) using the RNeasy kit purchased from Qiagen (Cat. No. 74104) according to the protocol provided by the vendor. Total RNA was extracted from. For each sample, total RNA was finally eluted with 50 μl of water. Using a cDNA synthesis kit with random primers (Promega Corporation catalog A3500), reverse the total RNA (9.5 μl of each sample) by placing the first cDNA strand in a 20 μl reaction according to the protocol provided by the vendor I copied it. The PCR reaction was set up using the following hi-fi PCR master mix manufactured by Roche Biochemical (Cat. No. 2 140 314) (Cat. No. 2 140 314).

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Both primers were synthesized by Invitrogen custom oligonucleotide and 2.5 O.D. D. Dissolved in water at a concentration of (260 nm) / ml. With this primer pair, the predicted PCR product for a particular H19 mRNA was 117 base pairs.

PCR reactions were performed in a Techne Genius ^™ heat cycle set (after initial denaturation at 94 ° C. for 3 minutes, denaturation at 94 ° C. for 30 seconds, followed by annealing and extension for 25 cycles at 60 ° C. for 30 seconds). The final extension was 72 ° C for 7 minutes. PCR products were fractionated by electrophoresis on a 2% agarose gel prepared in Tris borate buffer (80 volts, 2 hours), stained with ethidium bromide, and visualized by illumination with ultraviolet light. . Fluorescence photographs were taken with Kodak Image Station 440 CF.

  Amplification of cDNA from human fetal urinary bladder-derived epithelial cells yielded a clear band of PCR amplification product of the expected size (about 117 bp). No amplification product was observed when total RNA was used as a template. Amplification from H19 mRNA confirmed the RT-PCR product.

Example 4 Human Bladder Tissue Modeling
From Sprague-Dawley neonatal (24 hour) rats, the bladder mesenchyme was dissected and transferred to a dish containing 1% trypsin and incubated at 4 ° C. for 90 minutes. The medium was removed and the relics were then washed several times with DMEM containing 20% FBS to neutralize trypsin activity. The bladder epithelium and connecting mesenchyme were then further dissected under a dissecting microscope. To make tissue genetically engineered mesenchyme, a mesenchyme without connective epithelium was placed on the surface of a 0.4% agar plate containing DMEM containing 1% FBS. It should be noted that these short-term exposures of mesenchymal and tissue genetic recombinants to serum-containing media are not considered “incubated” in serum-containing media.

Primitive fetal human bladder-derived epithelial cells were collected by treatment with 0.5% collagenase / dispase. The bladder-derived epithelial cells were peeled off as a sheet. The cell sheet was then placed on the mesenchyme using sterile forceps and the tissue was incubated overnight at 37 ° C. in a humidified incubator with 95% air (5% CO ₂ atmosphere). On the next day, the tissue genetic recombinants were transplanted under the kidney capsule of CD1 nude mice.

  Eight weeks after transplantation, host mice were sacrificed, the tissues were collected and fixed overnight with 10% neutral formalin. The fixed tissue was dehydrated by using a series of graded aqueous ethanol solutions and cleaned with Histo-Clear solution. The tissue was then embedded in paraffin and cut to 6 μm. For basic staining, sections were dewaxed with Histo-Clear, rehydrated with graded ethanol solution, and stained with hematoxylin-eosin (H & E).

  In immunohistology, anti-human smooth muscle α-actin antibody, anti-human uroplakin antibody, anti-human cytokeratin 7 antibody, and anti-human cytokeratin 19 antibody were used. In the immunohistology of cytokeratin 7 and cytokeratin 19, an antigen retrieval protocol was performed. Briefly, the sections are soaked in Target Retrieval solution (Dako, Cat. No. 1699, diluted 1:10 from 10 × stock) and 30 minutes using microwave (Panasonic, NN-7523 model, 120V, 12.8A). Heated vigorously and then cooled to room temperature (about 25 ° C.). Antigen search was not performed for anti-human smooth muscle α-actin and uroplakin III.

Sections were blocked for 20 minutes with Milli-Q H ₂ O containing 3% H ₂ O ₂ and rinsed 3 times (2 minutes / wash) with 1 × PBS. The sections were then rounded using an Image ^™ pen (Vector Laboratories, catalog number H-4000) and washed again with 1 × PBS containing 0.5% Tween-20 for 2 minutes. The sections were further blocked with 1 × PBS (blocking buffer) containing 5% goat serum for 1 hour. Primary antibody was then added over the section (approximately 500 μl). The primary antibodies used were mouse anti-human smooth muscle α-actin antibody (Dako catalog number M0851, used without dilution), mouse anti-uroplatin III clone AU1 antibody (catalog number RDI-PR0651108, Research Diagnostics Inc., Flanders, New Jersey, 1: 500 dilution), mouse anti-human cytokeratin 7 antibody (Cat # 18-0234, Zymed, 1:50 dilution), and mouse anti-human cytokeratin 19NCL-CK19 (Cat # 100902, Novocastra, 1: 100 dilution) there were. Primary antibody incubation was performed overnight at 4 ° C.

  After overnight incubation, excess primary antibody was flushed and the sections rinsed 3 times (2 min / wash) with 1 × PBS containing 0.5% Tween-20. A secondary antibody (biotinylated goat anti-mouse immunoglobulin antibody (Cat. No. E0433, Dako) diluted 1: 500) was added (500 μl of each section) and incubated for 1 hour at room temperature. The sections are rinsed again 3 times (2 min / wash) with 1 × PBS containing 0.5% Tween-20 and according to the protocol provided by the manufacturer, Vectastein ABC Elite kit (Catalog No. PK-6100, Vector ) For further treatment. The slides were developed with a 1 mg / ml DAB solution (diaminobenzidine, Sigma, Catalog No. D-5905) containing 0.1 M NaAc (containing 0.015% hydrogen peroxide at pH 5.0). Finally, slides are washed with water, counterstained with hematoxylin (Cat. No. HXHHEGAL, American Master Tech Scientific) to identify nuclei, and graded ethanol (70%, 80%, 95%, 100%) (Harleco, Catalog No. 65347/85).

  The results are shown in FIG. 1 and FIG. FIG. 1a shows a section of H & E stained tissue gene recombinant. FIGS. 1b, 1c and 1d show α-actin, human cytokeratin 7 and human cytokeratin 19 staining, respectively. It should be noted that cytokeratin staining is limited to the epithelial layer on the inner surface of the cell lumen formed in the tissue recombinant. FIG. 2a shows staining with anti-human uroplakin antibody (FIG. 2b is a secondary antibody only control). As found normally, staining is limited to the cell luminal surface of the epithelial layer in the genetically modified in situ bladder epithelium. It should be noted that the anti-uroplakin III antibody stains only a portion of the fetal urinary bladder-derived epithelial cells, indicating the varying level of differentiation of the fetal urinary bladder-derived epithelial cells within the tissue genetically modified organism.

Example 5 Human Prostate Tissue Modeling
From Sprague-Dawley neonatal (birth) rats, seminal vesicle mesenchyme was dissected, transferred to a dish containing 1% trypsin and incubated at 4 ° C. for 90 minutes. The medium was removed and the relics were then washed several times with DMEM containing 20% FBS to neutralize trypsin activity. The seminal vesicle epithelium and connecting mesenchyme were then further dissected under a dissecting microscope. In order to prepare a tissue genetic recombinant, a mesenchyme without a connective epithelium was placed on the surface of a 0.4% agar plate containing FMEM-containing DMEM, and human fetal bladder epithelial inducible cells (Example 4) were prepared. Cultivated and collected as described in) and placed on mesenchyme and allowed to adhere overnight at 37 ° C. under humidified 95% air (5% CO ₂ ). It should be noted that these short-term exposures of mesenchymal and tissue genetic recombinants to serum-containing media are not considered “incubated” in serum-containing media.

  The tissue gene recombinant was transplanted under the kidney capsule of male SCID mice. Six months after transplantation, the host mice were sacrificed and the grafts were removed.

  As in Example 4, fixed tissues were treated and stained for prostate specific antigen by immunohistology. For immunohistology, sections were deparaffinized with histoclear and dehydrated in phosphate buffered saline through a continuous bath of ethanol. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 minutes. The tissue sections were then incubated overnight at 4 ° C. with an antibody against an antigen specific for the prostate (Dako corporation, California). Samples were washed with PBS and then incubated with secondary antibody (biotinylated goat anti-mouse antibody, dako corporation, California) followed by avidin-biotin complex. Staining was developed with diaminobenzidine (DAB). To remove excess DAB, the samples were extensively washed with water, dehydrated with graded alcohol, cleared with xylene, and mounted with a coverslip. As shown in FIG. 3, the results showed that bladder epithelial cells differentiated morphologically and biochemically into prostate epithelial cells when recombined with seminal vesicle mesenchyme.

  This application includes figures created in at least one color. Copies of this application with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

FIG. 1A shows a photomicrograph of a sample of human fetal urinary bladder-induced epithelial cells from passage 1 observed under a phase contrast microscope (100 ×, Nikon).

FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E and FIG. 1F show photomicrographs of samples of human fetal urinary bladder-induced epithelial cells from passage 1 stained with immunohistochemistry (IHC). FIG. 1B shows a negative control without the primary antibody. FIG. 1C shows cytokeratin 7 staining. FIG. 1D shows anti-human smooth muscle α-actin staining. FIG. 1E shows cytokeratin 19 staining. FIG. 1F shows uroplatin staining. The brown area is positive staining and the blue color is nuclear staining.
FIG. 2 shows a photomicrograph of a section from a tissue recombinant containing human fetal bladder-induced epithelial cells and rat bladder mesenchyme. FIG. 2A is a photomicrograph of hematoxylin and eosin (H & E) stained sections. FIG. 2B shows a section stained with an anti-human smooth muscle α-actin antibody. 2C and 2D show staining with antibodies specific for human cytokeratin 7 and human cytokeratin 19, respectively. Antibody binding is indicated by the deposition of a brown reaction product. FIG. 2E shows a section stained with anti-human uroplatin-III, and FIG. 3F shows a control (no primary antibody). Antibody binding (indicated by dark purple / black reaction product deposition) can be observed in FIG. 2E on the luminal surface. FIG. Differentiation of human bladder epithelial cell lines into prostate epithelial cells in tissue recombinants. FIG. 3A shows micrographs of paraffin sections (H & E staining) of epithelial cell sheets released from cultures of bladder epithelial cell lines prior to recombination with neonatal rat seminal vesicle mesenchyme. FIG. 3B shows micrographs of paraffin sections (H & E staining) of tissue recombinants of rat seminal vesicle mesenchyme grown under the kidney capsule of bladder epithelial cell lines and SCID mice for 6 months. A typical prostate structure is observed. FIG. 3C shows a photomicrograph of immunohistochemical staining of prostate specific antigen (PSA) in the same tissue recombinant as in FIG. 3B. Strong positive staining (brown) is observed in the epithelial cell lineage. The sections were counterstained with hematoxylin that stains cell nuclei in blue.

Claims (22)

A population of substantially pure human fetal urinary bladder-derived epithelial cells, wherein the cells have the ability to differentiate into bladder epithelium or prostate epithelium. The population of human fetal bladder-derived epithelial cells according to claim 1, wherein the cells are maintained in a serum-free medium. The population of human fetal bladder-derived epithelial cells according to claim 1, wherein the cell surface of the cells is substantially free of serum biomolecules. A tissue culture container comprising the population of human fetal urinary bladder-derived epithelial cells according to claim 1. A method of isolating a substantially pure population of human fetal urinary bladder-derived epithelial cells, the method comprising:
(A) microdissecting the origin of human fetal urinary bladder-derived epithelial cells;
(B) placing the bladder-derived epithelial cell origin in serum-free nutrient medium under culture conditions sufficient to maintain fetal bladder epithelial cells, wherein the serum-free medium comprises insulin, transferrin, α-tocopherol and aprotinin Including a nutritional process comprising:
(C) maintaining appropriate culture conditions sufficient to allow migration of the fetal bladder-derived epithelial cells from the bladder-derived epithelial cell origin in the serum-free nutrient medium;
(D) maintaining appropriate culture conditions sufficient to allow bladder-derived epithelial cells to form monolayer colonies; and (e) a substantially pure population of human fetal bladder-derived epithelial cells. Subculturing the monolayer colony to obtain,
Including the method. 6. The method of claim 5, wherein the serum-free nutrient medium further comprises progesterone, keratinocyte growth factor (KGF) and heregulin (HRG). Less than:
(A) microdissecting the origin of human fetal urinary bladder-derived epithelial cells;
(B) placing the bladder-derived epithelial cell origin in serum-free nutrient medium under culture conditions sufficient to maintain fetal bladder epithelial cells, wherein the serum-free medium comprises insulin, transferrin, α-tocopherol and aprotinin Including a nutritional process comprising:
(C) maintaining appropriate culture conditions sufficient to allow migration of the fetal bladder-derived epithelial cells from the bladder-derived epithelial cell origin in the serum-free nutrient medium;
(D) maintaining appropriate culture conditions sufficient to allow bladder-derived epithelial cells to form monolayer colonies; and (e) a substantially pure population of human fetal bladder-derived epithelial cells. Subculturing the monolayer colony to obtain,
A population of substantially pure human fetal urinary bladder-derived epithelial cells produced by a process comprising: The cell population according to claim 7, wherein the serum-free nutrient medium used in the step further comprises progesterone, keratinocyte growth factor (KGF) and heregulin (HRG). A method of providing an xenogenous source of immunogen, wherein said method comprises an effective amount of a large number of human fetal urinary bladder-derived epithelial cells according to claim 1 for inducing an immune response in said recipient. A method comprising the step of administering to said recipient. A method of eliciting an immune response in a heterologous recipient, said method comprising inducing an effective amount of a large number of human fetal bladder-derived epithelial cells according to claim 1 to induce an immune response in said recipient. A method comprising administering to an ent. A method of producing a population of differentiated human bladder epithelial cells from human fetal bladder-derived epithelial cells, said method being capable of supporting the growth of said human fetal bladder-derived epithelial cells in a non-human mammal recipient Administering a human fetal urinary bladder-derived epithelial cell according to claim 1 at a position within the ent, wherein the human fetal urinary bladder-derived epithelial cell is ex vivo and the human fetal urinary bladder-derived epithelial cell is human. A method that is recombined with mesenchymal tissue to affect differentiation into bladder epithelial cells. A human produced by a process comprising administering the human fetal urinary bladder-derived epithelial cell according to claim 1 to a location within a non-human mammal recipient capable of supporting the growth of human fetal urinary bladder-derived epithelial cells. A population of human bladder epithelial cells differentiated from fetal bladder-derived epithelial cells, wherein the human fetal bladder-derived epithelial cells are ex vivo and affect the differentiation of the human fetal bladder-derived epithelial cells into human bladder epithelial cells A population of human bladder epithelial cells that can be recombined with mesenchymal tissue. A method of producing a population of differentiated human prostate epithelial cells from human fetal urinary bladder-derived epithelial cells, wherein the method is at a location within a non-human mammal recipient that can support the growth of the human fetal urinary bladder-derived epithelial cells. And administering the human fetal urinary bladder-derived epithelial cell according to claim 1, wherein the human fetal urinary bladder-derived epithelial cell is ex vivo and the human fetal urinary bladder-derived epithelial cell is transferred to a human prostate epithelial cell. A method that is recombined with mesenchymal tissue that can affect differentiation. A human produced by a process comprising administering the human fetal urinary bladder-derived epithelial cell according to claim 1 to a location within a non-human mammal recipient capable of supporting the growth of human fetal urinary bladder-derived epithelial cells. A population of human prostate epithelial cells differentiated from fetal urinary bladder-derived epithelial cells, wherein the human fetal urinary bladder-derived epithelial cells are ex vivo and affect the differentiation of the human fetal urinary bladder-derived epithelial cells into human prostate epithelial cells A population of human prostate epithelial cells that can be recombined with mesenchymal tissue. A method of producing a human bladder tissue model in a non-human mammal recipient, said method being at a location within the non-human mammal recipient that can support the growth of said human fetal bladder-derived epithelial cells. A step of administering the human fetal urinary bladder-derived epithelial cell according to Item 1, wherein the human fetal urinary bladder-derived epithelial cell is used for ex vivo differentiation of the human fetal urinary bladder-derived epithelial cell into a human urinary bladder epithelial cell. A method that is recombined with mesenchymal tissue so that it can be affected. A human produced by a process comprising administering the human fetal urinary bladder-derived epithelial cell according to claim 1 to a location within a non-human mammal recipient capable of supporting the growth of human fetal urinary bladder-derived epithelial cells. A bladder tissue model, wherein the human fetal bladder-derived epithelial cells are recombined with mesenchymal tissue so as to be able to affect the differentiation of the human fetal bladder-derived epithelial cells into human bladder epithelial cells ex vivo, Human bladder tissue model. A method of producing a human prostate tissue model in a non-human mammal recipient, said method being at a location within the non-human mammal recipient capable of supporting the growth of said human fetal bladder-derived epithelial cells. The method includes the step of administering the human fetal urinary bladder-derived epithelial cell according to Item 1, wherein the human fetal urinary bladder-derived epithelial cell is used to differentiate the human fetal urinary bladder-derived epithelial cell into a human prostate epithelial cell ex vivo. A method that is recombined with mesenchymal tissue so that it can be affected. 2. A process comprising administering the human fetal bladder-derived epithelial cell of claim 1 to a location within the recipient that can support the growth of human fetal bladder-derived epithelial cells in a non-human mammal recipient. A human prostate tissue model produced, wherein the human fetal bladder-derived epithelial cells are ex vivo and mesenchymal tissue that can affect the differentiation of the human fetal bladder-derived epithelial cells into human prostate epithelial cells A human prostate tissue model that is recombined. A method for providing a source of biological components specific to a bladder-derived tissue for pharmaceutical development of at least one drug, the method comprising the following: Isolating a population of cells and using the bladder-derived epithelial cells or any cell portion of the cells as a target for a drug under development. A method for providing a source of human fetal urinary bladder-derived epithelial cells for pharmaceutical development of at least one drug, the method comprising the following: And using the human fetal urinary bladder-derived epithelial cells as a target for a drug under development. A method of providing a source of nucleic acid or protein for a bioassay comprising isolating the nucleic acid or protein from human fetal bladder-derived epithelial cells according to claim 1 and one or more Using the nucleic acid or protein in a bioassay as a major component of. A method for providing a source of human fetal urinary bladder-derived epithelial cells for a bioassay comprising the step of providing a population of human fetal urinary bladder-derived epithelial cells according to claim 1, and a bioassay. Using the human fetal urinary bladder-derived epithelial cells.

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